Amine Detection Method and Materials

ABSTRACT

Compounds linked to a solid support through a divalent linker moiety are disclosed. In particular, compounds such as 1-hydroxybenzotriazole-6-carboxylic acid are directly linked to the support under mild conditions (i.e., in aqueous or organic solvents at neutral pH and at room temperature). The polymer bound 1-hydroxybenzotriazole-6-carboxylic acid can be used for the derivatization of amines as well as for single step amino group modification of proteins, peptides, and amines via acylation or sulfonylation reactions. A flow through device and method for the single step amino group modifications of proteins, peptides, and amines is disclosed. Also disclosed is a flow through device for the detection of amines in a sample. Additionally, a device and method for the detection of amines in a sample using 1-hydroxybenzotriazole-6-carboxylic acid are disclosed. In a preferred embodiment, the device is used to detect the presence of amines in a spoiled meat product. Diagnostic kits for detecting the presence of amines are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of application Ser.No. 09/983,743 filed Oct. 25, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to polymer bound compounds suchas 1-hydroxybenzotriazole-6-carboxylic acid (P-HOBT) and uses thereof.In particular, the present invention relates to a device for thesingle-step amino group modification of proteins, peptides, and aminesvia acylation reactions. The present invention also relates to a methodof detecting the presence of amines in a sample using polymer boundcompounds such as 1-hydroxybenzotriazole-6-carboxylic acid.

2. Description of the Related Art

Combinatorial chemistry has become one of the major tools in the searchfor new lead compounds in pharmaceutical drug discovery (1-3). As theuse of combinatorial chemistry increases, much effort has gone into thedevelopment of new synthetic methods for the highly efficientpreparation of chemical libraries. One of the results of this researchhas been an increase in the development and use of solid-phase organicsynthesis techniques.

Two general protocols have been used in solid-phase organic synthesis:(i) the use of polymers as a scaffold upon which to build the desiredmolecule in a multi-step sequence, or (ii) the use of apolymer-supported reagent to mediate a single synthetic step. The use ofpolymers as templates for building complex molecules has been exploitedin well-known applications such as solid-phase peptide synthesis (4) andoligonucleotide synthesis (5).

Beyond these technologies, a variety of organic reactions have beensuccessfully carried out in the solid phase (3, 5-11). Moreover, thismethodology has been used for the synthesis of small heterocyclicorganic molecules, including benzodiazepines, diketopiperazines andhydantoins (12-14).

Alternately, the use of polymer supported acylating agents as catalystshas been particularly useful for the formation of amide bonds, as wellas ester formation. These immobilized reagents include nitrophenol(15-17), N-hydroxysuccinimide (NHS) (18), 1-hydroxybenzotriazole (HOBT)(19-21), carbodiimide (22, 23), and triphenylphosphine (24, 25). Ofthese polymer supported acylating agents, polymer supported HOBT(P-HOBT) has been the most useful. P-HOBT has been used as a highlyreactive N-acylating agent for the formation of peptide bonds (19-21)and simple amides (26, 27). P-HOBT has also been utilized for thesynthesis of medium-ring lactams from linear ω-amino acid precursors(28), as an acylating agent for the synthesis of NHS esters (29), andfor the carbamate protection of primary and secondary amines (30).Excluding solid-phase peptide synthesis and organic synthesis, currentprotein, peptide, and amine modifications are carried out in solutionphase reactions which involves subsequent work-up and/or purification ofthe product after the reaction.

In the work of Fridkin and Patrchornik (19), the immobilized HOBTreagent was synthesized by the direct functionalization of polystyreneutilizing 4-chloro-3-nitrobenzyl alcohol under Friedel-Craftsconditions. The HOBT moiety was formed by treatment of the polymer withhydrazine monohydrate and subsequent cyclization under acidic conditionsusing known methods (31). Although this method has been utilized for thepreparation of P-HOBT, it is limited to polystyrene (aryl) basedpolymers, and is not always reproducible due to the Friedel-Craftsreaction, the synthesis of P-HOBT was improved by the initial couplingof 4-chloro-3-nitrobenzenesulfonyl chloride to an aminomethylatedpolystyrene polymer, followed by reaction with hydrazine monohydrate andcyclization under acidic conditions (26). The polymer is highly activedue to the presence of the electron withdrawing sulfonyl group in thebenzene ring, and can by synthesized in a reproducible manner. Adisadvantage of this approach is the two step method of HOBT cyclizationafter addition of the sulfonyl chloride to the polymer, utilizingconditions which may not be compatible for the derivatization of allsolid supports. This limits the choice of solid supports used toimmobilize the HOBT and requires the adaptation of an application toutilize a polymer support that can be modified using the heretoforeknown method of coupling 4-chloro-3-nitrobenzoic acid to the polymericsupport, followed by the two step formation of HOBT.

Thus, before the present invention, there were no known effective meansof directly immobilizing 1-hydroxybenzotriazole or a derivative thereofto a solid support under mild conditions, i.e., in organic or aqueoussolvents at neutral pH and at room temperature. Additionally, there wereno known methods of using immobilized 1-hydroxybenzotriazole orderivatives thereof for self-contained, single step modifications ofamino groups on proteins, peptides, and amines, or to using1-hydroxybenzotriazole-6-carboxylic acid to effectively and efficientlydetect the presence of amines in a sample.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a compound such as1-hydroxybenzotriazole-6-carboxylic acid linked to a solid support.

It is another object of the present invention to provide a flow-throughdevice for the one-step modification of proteins, peptides, and aminogroups via acylation reactions.

It is yet another object of the present invention to provide a method ofdetecting the presence of amines in a sample.

It is a further object of the present invention to provide a device fordetecting the presence of amines in a sample.

It is yet another object of the present invention to provide aflow-through device to detect the presence of amines.

It is yet another object of the present invention to provide adiagnostic kit to indicate the spoilage of food products.

It is a further object of the present invention to provide a meatpackaging sensor to indicate the spoilage of food products.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the two step formation of1-hydroxybenzotriazole-6-carboxylic acid.

FIG. 2 is a schematic of the single step formation of polymer bound1-hydroxybenzotriazole-6-carboxylic acid.

FIGS. 3 a-3 f are schematics of general reactions for the formation oflactones, amides, lactams, carbamates, esters, and sulfonamides usingpolymer bound 1-hydroxybenzotriazole-6-carboxylic acid.

FIG. 4 is a schematic of a general acylation reaction using polymerbound 1-hydroxybenzotriazole-6-carboxylic acid.

FIG. 5 a is an illustration of a flow-through cartridge device accordingto one embodiment of the present invention.

FIG. 5 b is an illustration of a flow through membrane device accordingto one embodiment of the present invention.

FIG. 5 c is an illustration of a flow through syringe device accordingto one embodiment of the present invention.

FIG. 6 a is an illustration of the meat packaging sensor according toone aspect of the present invention showing that the food is safe forconsumption.

FIG. 6 b is an illustration of the meat packaging sensor according toone aspect of the present invention showing that the food is not safefor consumption.

FIG. 7 is an illustration of the wafer sensor device according to oneembodiment of the present invention.

FIG. 8 is an illustration of the dye activated cellulose surface of thewafer sensor device according to one embodiment of the presentinvention.

FIG. 9 a is an illustration of the mode of action of the wafer sensordevice according to one embodiment of the present invention when themeat product is fresh.

FIG. 9 b is an illustration of the mode of action of the wafer sensordevice according to one embodiment of the present invention when themeat product is spoiled.

FIG. 10 is an illustration of the general chemistry involved in therelease of the dye in the wafer sensor device according to oneembodiment of the present invention.

FIG. 11 is an illustration of the amine diagnostic kit device accordingto one embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1, 1-hydroxybenzotriazole-6-carboxylic acid is formedin two steps from 4-chloro-3-nitrobenzoic acid. In the first step,4-chloro-3-nitrobenzoic acid and hydrazine monohydrate are heated atreflux in ethanol to yield a crude precipitate. In the second step, thecrude precipitate is dissolved in water and acidified with HCl to pH 1to yield 1-hydroxybenzotriazole, a peach colored precipitate.Confirmation of the compound can be conducted by NMR spectroscopy andhigh resolution mass spectroscopy (HRMS), as well as other detectionmethods determined by one of skill in the art.

Current methods for the preparation of polymer supported1-hydroxybenzotriazole (“P-HOBT”) rely on the ability to incorporate aderivative of 4-chloro-3-nitrobenzoic acid on the polymeric support,followed by the addition of hydrazine to the polymer in ethanol atreflux (80° C.) and subsequent cyclization under acidic conditions(e.g., HCl, pH 1) to yield the 1-hydroxybenzotriazole moiety. Thisapproach is disadvantageous in that the two step process for HOBTcyclization after the addition of 4-chloro-3-nitrobenzoic acid to thepolymer limits the choice of solid supports used to immobilize the HOBTmoiety.

On the other hand, 1-hydroxybenzotriazole-6-carboxylic acid has theability to link directly to a solid support under very mild conditions.The phrase “mild conditions” as used herein indicates that the reactionsare run in organic or aqueous solvents at neutral pH and at roomtemperature. The use of 1-hydroxybenzotriazole-6-carboxylic acid greatlyexpands the choice of supports from that which can be utilized when HOBTalone is linked to a support.

Furthermore, 1-hydroxybenzotriazole-6-carboxylic acid can be used forthe efficient and easy immobilization of the HOBT moiety on solidsupports that contain amino groups, hydroxyl groups, or other functionalgroups known to one of skill in the art to form stable adducts uponreaction with a carboxylic acid under mild conditions. For example, bylinking the 1-hydroxybenzotriazole-6-carboxylic acid directly to a solidsupport with an amino group, the HOBT moiety is coupled to the solidsupport through an electron withdrawing amide linkage. As a result, thereactivity of HOBT as an acylating agent is increased.

Alternatively, derivatives of 1-hydroxybenzotriazole-6-carboxylic acidmodified at the carboxylate moiety to include another functionality suchas a sulfhydryl group may be used to link the HOBT moiety to a solidsupport. Additionally, 1-hydroxybenzotriazole-6-carboxylic acid andderivatives thereof at the carboxylate moiety can also be used toincorporate 1-hydroxybenzotriazole functionality in other organicmolecules that are not polymer based.

Although the use of 1-hydroxybenzotriazole-6-carboxylic acid is apreferred embodiment, other acylating agents would function in a similarfashion. Suitable acylating agents are substituted aromatic residues ofthe following general formula:

in which R′ is a mono—or polyaromatic ring, which may include one ormore heteroatoms, which is capable of forming activated acylating andsulfonylating agents. Examples include, but are not limited to,2,3,5,6-tetrafluoro-4-hydroxybenzoic acid, 4-hydroxy-3-nitrobenzoicacid, and 6-hydroxynicotinic acid, and2-hydroxy-1,3-dioxoisoindole-5-carboxylic acid. Alternatively, R′ may beany suitable non-aromatic acylating compound, including derivatives ofN-hydroxy succinimide, such as:

The single step linking of the 1-hydroxybenzotriazole-6-carboxylic acidto a polymer is shown in FIG. 2. As shown in FIG. 2, the aminoalkylatedpolymer is first suspended in N,N-dimethylformamide (DMF).1-Hydroxybenzotriazole-6-carboxylic acid and dicyclohexylcarbodiimide(DCC) or diisopropylcarbodiimide (DIC) is then added to the polymersuspension. After the reaction is complete, the polymer bound HOBT isfiltered and washed using DMF, dichloromethane (CH₂Cl₂), and methanol(MeOH). The derivatized polymer is treated with a large excess ofisopropylamine, followed by rigorous washing to provide the free HOBTreagent. The polymer supported HOBT retains its high reactivity, and theresulting electron withdrawing amide linkage enhances the reactivity ofthe immobilized HOBT moiety as a leaving group.

The choice of solid supports depends on the desired chemistry to beattempted with the polymer bound 1-hydroxybenzotriazole-6-carboxylicacid. For example, the use of the HOBT moiety immobilized on Merrifieldtype polystyrene (PS) resins, whose degree of swelling varies greatlydepending on the solvent, would be appropriate for reactions in organicsolvents, but would not be appropriate under aqueous conditions. For thederivatization of peptides or proteins, a solid support which has theability to be used in aqueous and organic solvents without swelling orcontraction is desirable. Polystyrene and polystyrene/polyethyleneglycol polymers are limited to reactions in organic solvents due totheir poor and limited swelling properties in water. Additionally,silica and glass bead based solid supports can be used in aqueous ororganic solvents without swelling or contraction. Supports suitable forthe immobilization of the 1-hydroxybenzotriazole-6-carboxylic acid wouldbe easily identified by one of skill in the art and include supportssuch as polystyrene, polystyrene/polyethylene glycol graft copolymers,silica gels, glass beads, controlled pore glass, agarose, sepharose,cellulose, chitosan, polyacrylonitrile, polyurethane, polypropylene,polyvinyl alcohol, polymethacrylate, polyacrylamide, polysulfone andmodified derivatives thereof.

Immobilized 1-hydroxybenzotriazole-6-carboxylic acid can be used inliquid chromatography for the derivatization (on-line and off-line) ofnaturally occurring amines for improved separation and identificationproperties. Amines are widely found in nature. For example, biogenicamines are present in living cells and in food products. A number ofnaturally occurring primary and secondary amines can be found in meatand fish as it spoils (e.g., histamine, putrescine, cadaverine, methylamine), tobacco smoke (e.g., methylamine, dimethylamine, pyrrolidine),and beer (e.g., ethylamine, isoamylamine, dibutylamine). Coniine (i.e.,2-propylpiperidine, hemlock poison) is a simple non-chromophoric cyclicamine compound. Other naturally occurring, non-chromophoric aminesinclude glucosamine, galactosamine, mannosamine, and heparin. Althoughamines are generally easy to separate, the lack of a chromophore intheir structure makes amines difficult to detect. To overcome thisproblem, P-HOBT is used for the one-step derivatization of amines with adetectable agent, including, but not limited to chromophoric agents(e.g., FD&C Red 3, or an azo dye such as DABCYL, DABSYL), fluorogenicagents (e.g., pyrene, fluorescein, lucifer yellow, BODIPY, rhodamine,DANSYL, EDANS, or derivatives thereof), radioactive agents (e.g., ³H,¹⁴C, ¹²⁵I, ³⁵S, or ³²P), and electrochemical active agents which mayinclude a thiol or catechol moiety. The derivatization may occur in anaqueous environment or in an organic solvent. The one-stepderivatization of amines with chromophoric or fluorogenic agents usingP-HOBT allows for the specific and sensitive determination of amines,such as with UV/V is and fluorescence detection.

Additionally, immobilized 1-hydroxybenzotriazole-6-carboxylic acid canbe used for amino group modifications of proteins, peptides, and aminesvia acylation and sulfonylation reactions in either organic or aqueousenvironments. These modifications include the formation of amides,carbamates, and sulfonamides. Moreover, immobilized1-hydroxybenzotriazole-6-carboxylic acid can be used for themodification of amines to lactams, and the conversion of hydroxyl groupsto esters and lactones. General reactions for the formation of lactones,amides, lactams, carbamates, esters, and sulfonamides using polymerbound 1-hydroxybenzotriazole-6-carboxylic acid are shown in FIGS. 3 a-3f. Applications of these chemistries include radiolabeling (e.g., ¹²⁵I,³H, ¹⁴C, ³⁵S, or ³²P), chromophoric labeling, fluorescent labeling,affinity labeling (e.g., biotin), activation for covalent crosslinking(e.g., maleimido or azido group), and amine protection as carbamates(e.g., Fmoc, Cbz, Boc) or sulfonamides (e.g. tosylates). P-HOBT isideally suited for this type of chemistry since the derivatizing reagentis immobilized, and once the reaction is complete, all that exists inthe solution is the product.

The general acylation reaction for polymer bound1-hydroxybenzotriazole-6-carboxylic acid is shown in FIG. 4. For ease ofsynthesis and potential automation, acylation reactions are typicallycarried out in 8 ml Extract-Clean™ solid phase extraction tubes equippedwith a disposable inlet cap and a one-way stopcock on the outlet.Reactions are mixed by gentle rocking and filtration is carried outusing a 12-port solid phase extraction manifold connected to a wateraspirator. Initially, the polymer-bound ester (R=alkyl, aryl) orcarbonate (R=alkoxy) is formed by the addition of the acid anhydride,acid chloride or the desired chloroformate reagent and pyridine to theimmobilized HOBT group, or by coupling the free carboxylic acid to thepolymer using DCC or DIC as a catalyst. After washing the activatedresin thoroughly to remove side products, the resin is suspended insolvent containing 0.8 equivalents of amine (or other nucleophile) basedon polymer activity. The reaction mixture is then rocked at roomtemperature. Filtration and subsequent concentration of the filtrateyields the desired product. The recovered polymer bound HOBT is fullyrecoverable and recyclable.

The general suflonylation reaction for polymer bound1-hydroxybenzotriazole-6-carboxylic acid is carried out in an analogousfashion to the acylation reaction. For ease of synthesis and potentialautomation, suflonylation reactions are typically carried out in 8 mlExtract-Clean™ solid phase extraction tubes equipped with a disposableinlet cap and a one-way stopcock on the outlet. Reactions are mixed bygentle rocking and filtration is carried out using a 12-port solid phaseextraction manifold connected to a water aspirator. Initially, thepolymer-bound sulfonate ester (R=alkyl, aryl) is formed by the additionof the sulfonyl chloride and pyridine to the immobilized HOBT group.After washing the activated resin thoroughly to remove side products,the resin is suspended in solvent containing 0.75 equivalents of aminebased on polymer activity. The reaction mixture is then rocked at roomtemperature. Filtration and subsequent concentration of the filtrateyields the desired product. The recovered polymer bound HOBT is fullyrecoverable and recyclable.

The immobilized HOBT can be used for amino group modification ofproteins, peptides, and amines via acylation reactions and sulfonylationreactions such as in batch reactions or in a flow-through device such asa cartridge containing a polymer-bound reagent (see FIG. 5 a) ormembrane-bound reagent (FIG. 5 b), syringe device (see FIG. 5 c), orcolumn (not shown). The flow through device is not limited, and includesother flow through devices readily determined by one of skill in theart. A variety of modifications may be performed depending on theacylating agent used, including radiolabeling, fluorescent labeling,affinity labeling, activation for covalent crosslinking, and amineprotection. The immobilized HOBT can be used to modify amines with adetectable agent such as chromophoric agent, a fluorogenic agent, aradioactive agent, or an electrochemical agent. Suitable examples ofdetectable agents are set forth above, but would be easily determined byone of skill in the art. In general, the polymer bound1-hydroxybenzotriazole-6-carboxylic acid is self-contained in aprepackaged, single use, readily disposable flow-through device. Theflow-through device can be prepared from microgram to multi-gram scale(e.g. up to 10 g).

In one embodiment, the polymer bound 1-hydroxybenzotriazole-6-carboxylicacid is pre-packaged in a flow-through polypropylene cartridge with theinlet designed for the attachment of a luer tip/syringe device and theoutlet is either designed for attachment to a luer tip or is a luer tip.Preferably, the 1-hydroxybenzotriazole-6-carboxylic acid is linked to asolid support. Examples of suitable supports include polystyrene,polystyrene/polyethylene glycol graft copolymer, silica gel, glassbeads, controlled pore glass, agarose, sepharose, a solid polymer havinga primary amine, a solid polymer having a secondary amine, cellulose,polypropylene, polyurethane, chitosan, polyacrylonitrile, polysulfone,polymethacrylate, polyacrylamide, polyvinyl alcohol and modifiedderivatives thereof. A luer tip syringe (e.g., a 2.5 ml polypropylenesyringe) is attached to the inlet.

Preparation of the device for reaction occurs by wetting the device withthe desired solvent using the syringe. The substrate to be modified isdiluted in the wetting solvent and is then added to the device throughthe syringe. Acylation takes place by allowing the substrate solution topass through the device into a collection vessel which can be attacheddirectly to the device or placed below the device. The substrate canpass through the device by gravity, it can be forced through the devicesuch as by with the syringe plunger, or it can be pulled through by theapplication of vacuum to the outlet. The modified substrate can beutilized as is as it exits the device, or it can be isolated by removalof the solvent. Once used, the device is thrown away. Thus, there isprovided a means for a single-step modification of theprotein/peptide/amine group via an acylation or sulfonylation reaction.Since no soluble reagents are added to the substrate solution, nopurification is required. The advantages of such a device include easeof use, facile disposal of spent reagent (i.e., the cartridge can bethrown away intact), no work-up or product purification needed,cartridges can be prepared from microgram to gram scale, minimal amountsof solvent are needed, and the device is cost effective.

Not only can polymer bound 1-hydroxybenzotriazole-6-carboxylic acid beused for acylation reactions, etc., it can be used to detect amines in asample via amide or sulfonamide derivatization. In one embodiment, thepolymer bound 1-hydroxybenzotriazole-6-carboxylic acid bears a dye, or aspecific color or signal, as an activated ester or sulfonate ester.Other linkages and indicators can be easily determined by one of skillin the art, and are within the scope of the present invention. The HOBTbearing the color indicator or signal is bound to a porous membranewhich allows the unrestricted passage of amines. The1-hydroxybenzotriazole-6-carboxylic acid can be bound to the membrane byamide bond formation or can be directly bound via an ester linkage.Underlaying the HOBT bound membrane is a porous membrane having apre-designated molecular weight cutoff (MWCO) which permits smallmolecules such as amines to pass, but will not allow intact cells,proteins, or other contaminants to pass. Overlaying this amine-passingmembrane layer is a coating through which unbound dye may diffuse. Thiscoating layer may be overlayed with a masking layer, which limits theportions of the coating layer that are visible to the naked eye. Whenamines are free in the sample, they pass through the porous membrane andreact with the immobilized 1-hydroxybenzotriazole-6-carboxylic acid,causing the release of the dye. The dye then diffuses through thecoating layer located over the amine-passing membrane, causing a colorchange on the coating layer on any portion not covered by the maskinglayer, thus indicating the presence of amines. The intensity of thecolor change is proportional to the total amine concentration in thesample.

In another embodiment, the polymer bound1-hydroxybenzotriazole-6-carboxylic acid bears a dye, or a specificcolor or signal, as an activated ester or sulfonate ester. Otherlinkages and indicators can be easily determined by one of skill in theart, and are within the scope of the present invention. The HOBT bearingthe dye, color indicator or signal is bound to a solid support which isfurther contained in a porous bag (e.g. tea bag) or in a porous, butrigid material (e.g. test strip). The1-hydroxybenzotriazole-6-carboxylic acid can be bound to the solidsupport by amide bond formation. The “tea bag” or “test strip” HOBTreagent is contained in a transparent reaction vessel (e.g. test tube,or cuvette) to which is added reaction media (buffer) suitable for theefficient acylation/sulfonylation of amines by HOBT.

After a sample containing amines is added to the reaction vessel, thereaction vessel is closed and agitated. During agitation, the samplepasses through the “tea bag” or “test strip” and free amines present inthe sample react with the immobilized1-hydroxybenzotriazole-6-carboxylic acid, releasing the dye. The dyethen diffuses into the bulk solution, causing a color change throughoutthe reaction vessel, thus indicating the presence of amines. The HOBTreagent may then be removed from the reaction vessel to stop any furtherreaction from occurring. The intensity of the color change isproportional to the total amine concentration in the sample, providing aqualitative analysis for amine content. An aliquot of the buffersolution may be further analyzed by known techniques (e.g. highperformance liquid chromatography) for quantitative identification ofthe structure and concentration profile of the amines present in thesample.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES 1. Preparation of Polymer-Supported HOBT

Formation of 1-hydroxybenzotriazole-6-carboxylic Acid

A solution of 4-chloro-3-nitrobenzoic acid (5.047 g, 25.60 mmol) andhydrazine monohydrate (25 ml) in 250 ml of 95% ethanol was heated atreflux for 5.5 hours. At this time, the solution turned dark orange anda precipitate formed. The suspension was then cooled to 25° C. andfiltered. Next, the orange precipitate was washed with cold ethanol. Theprecipitate was dissolved in 500 ml of water and the pH was adjusted to1 using concentrated HCl. A peach colored precipitate formed during thistime. The precipitate was collected by filtration, washed with water,and dried in vacuo to yield 3.153 g (69%).

¹H NMR: (300 MHz, DMSO-d₆) ä 7.92 (dd, 1H, J=0.9, 8.7 Hz, ArH ₆), 8.07(d, 1H, J=8.7 Hz, ArH ₅), 8.23 (s, 1H, ArH ₈), 13.6 (bs, 1H, COOH); ¹³CNMR: (CDCl₃) a 111.23, 112.354, 119.79, 118.77, 124.35, 125.33, 127.60,129.70, 144.54, 166.; HRMS calculated for C₇H₆O₃N₃ 180.04091 [M+1]⁺,found 180.04041 [M+1]⁺.

Polymer-Supported 1-hydroxybenzotriazole (P-HOBT 1)

Aminomethyl polystyrene (0.5010 g, 0.90 mmol/g NH₂, 0.451 mmol, Sigma,1% DVB crosslinked, 200-400 mesh) was washed with DMF (3×5 ml), MeOH(3×5 ml) and DMF (3×5 ml). The polymer was suspended in DMF and 0.2418 g(1.350 mmol, 2.99 equiv) of 1-hydroxybenzotriazole-6-carboxylic acid wasadded. The suspension was rocked for 10 minutes. DCC (0.2968 g, 1.438mmol, 3.19 equiv) was then added. The suspension was subsequentlystirred for 1.25 hours. At this time, the polymer was filtered with DMF(3×5 ml), CH₂Cl₂ (3×5 ml), DMF (3×5 ml), MeOH (3×5 ml), DMF (3×5 ml) andMeOH (3×5 ml), and aspirated for 15 minutes. The crude activated polymerwas then suspended in CH₂Cl₂ and isopropylamine (0.26 g, 0.38 ml, 4.4mmol) was added. The suspension was rocked for 1.25 hours, filtered withDMF (3×5 ml), CH₂Cl₂ (3×5 ml), DMF (3×5 ml) and MeOH (3×5 ml), andaspirated for 15 minutes to yield 0.569 g of P-HOBT.

Assay of Hydroxyl Group Content in P-HOBT 1

Acetic anhydride (0.206 g, 0.190 ml, 2.02 mmol) and pyridine (0.235 g,0.240 ml, 2.97 mmol) was added to a suspension of P-HOBT (0.506 g) inCH₂Cl₂ (5 ml). The suspension was subsequently rocked for 30 minutes at25° C. At this time, the polymer was filtered, washed with CH₂Cl₂ (3×5ml), DMF (3×5 ml), CH₂Cl₂ (3×5 ml), and anhydrous Et₂O (3×5 ml). Thepolymer was resuspended in CH₂Cl₂ (5 ml) and isopropylamine (0.278 g,0.400 ml, 4.70 mmol) was added. Next, the suspension was rocked at 25°C. for 4 hours. The polymer was then filtered and washed with CH₂Cl₂(4×5 ml). The filtrate and washings were combined and concentrated toyield 0.0121 g n-isopropyl acetamide as a clear oil. This gave anactivity of 0.592 mmol/g for P-HOBT.

¹H NMR (300 MHz, CDCl₃): ä 1.15 (d, 6H, J=9.0 Hz, CH (CH ₃)₂), 1.95 (s,3H, CH ₃CO), 4.03-4.08 (m, 1H, J=7.2 Hz (CH₃)₂CH), 5.45 (bs, 1H, NH);¹³C NMR (CDCl₃): ä 22.74 (C(CH₃)₂), 23.49 (CH₃CO), 41.35 (C(CH₃)₂),169.13 (CH₃ CO).

P-HOBT on Other Solid Supports

The method above was used to prepare P-HOBT on a variety of solidsupports as shown in Table 1.

TABLE 1 P-HOBT Activity —NH₂ HOBT P-HOBT Polymer activity activity 1Aminomethyl polystyrene 0.9 mmol/g 0.59 mmol/g (1% DVB crosslinked,200-400 mesh, Sigma Chemical Co.) 2 ArgoGel-NH₂ (120-230 μm, ArgonautTechnologies, Inc.) 0.42 mmol/g 0.25 mmol/g 3 ArgoGel-NH₂ (120-230 μm,Argonaut Technologies, Inc.) 0.42 mmol/g 0.39 mmo/g 4 3-aminopropylsilica gel (Aldrich Chemical Co.) 1 mmol/g 0.45 mmol/g 5 3-aminopropylsilica gel (Aldrich Chemical Co.) 1 mmol/g 0.45 mmol/g 6 3-aminopropylsilica gel (Aldrich Chemical Co.) 1 mmol/g 0.49 mmol/g 7 aminopropylcontrolled pore glass beads 0.14 mmo/g 0.13 mmol/g (170Å, 200-400 mesh,Sigma Chemical Co.) 8 3-aminopropyl controlled pore glass beads 0.57mmol/g 0.145 mmol/g (350Å, 200-400 mesh. CPG, Inc.) 9 3-aminopropylcontrolled pore glass beads 0.57 mmol/g 0.135 mmol/g (350Å, 200-400mesh. CPG, Inc.)

All acylation and sulfonylation reactions utilizing P-HOBT were carriedout in 4 mL or 8 mL Extract-Clean™ solid phase extraction tubes (fromAlltech Associates, Inc.), equipped with a disposable inlet cap and aone-way stopcock on the outlet. Reactions were mixed by gentle rocking.Filtration was carried out using a 12-port solid phase extractionmanifold connected to a water aspirator.

2. Amide Formation Using P-HOBT

N-benzylacetamide

To a suspension of P-HOBT 1 (0.212 g, 0.125 mmol) in CH₂Cl₂ (5 mL) wasadded acetic anhydride (0.216 g, 0.200 mL, 2.12 mmol, 17 equiv.) andpyridine (0.234 g, 0.240 mL, 2.96 mmol, 24 equiv.). The suspension wassubsequently rocked for 60 minutes at 25° C. The polymer was thenfiltered, washed with CH₂Cl₂ (3×5 mL), NMP (3×5 mL), CH₂Cl₂ (3×5 mL),and anhydrous Et₂O (3×5 mL). The polymer was re-suspended in CH₂Cl₂ (5mL) and benzylamine (0.0098 g, 0.010 mL, 0.91 mmol, 0.73 equiv. based onP-HOBT 1) was added. The suspension was rocked at 25° C. for 4 h. Thepolymer was then filtered and washed with CH₂Cl₂ (4×5 mL). The filtrateand washings were combined and concentrated to yield 0.014 g (100%) ofN-benzylacetamide as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 2.01 (s, 3H, C(O)CH ₃), 4.41 (s, 1H, ArCH),4.42 (s, 1H, ArCH), 5.90 (bs, 1H, NH), 7.29 (m, 5H, ArH); LRMScalculated for C₉H₁₁NO [M^(+] 149,) found [M⁺] 149.

3. Carbamate Formation Using P-HOBT

N-FMOC-benzylamine

To a suspension of P-HOBT 5 (1.0 g, 0.49 mmol) in CH₂Cl₂ (5 mL) wasadded fluorenylmethoxychyloroformate (0.350 g, 1.35 mmol, 3.0 equiv.)and pyridine (0.147 g, 0.150 mL, 1.86 mmol, 4.1 equiv.). The suspensionwas subsequently rocked for 60 minutes at 25° C. The polymer was thenfiltered and washed with CH₂Cl₂ (3×5 mL), MeCN (3×5 mL), and MeOH (3×5mL). The polymer was re-suspended in CH₂Cl₂ (5 mL), followed by theaddition of benzylamine (0.036 g, 0.037 mL, 0.34 mmol, 0.75 equiv. basedon P-HOBT 5). The suspension was rocked at 25° C. for 22 h. The polymerwas then filtered and washed with CH₂Cl₂ (2×5 mL). The filtrate andwashings were combined and concentrated to yield 0.084 g (76%) ofN-FMOC-benzylamine as a solid. LRMS calculated for C₁₉H₂₂NO₂ [M⁺] 329,found [M+1]⁺ 330.

4. Sulfonamide Formation Using P-HOBT

N-benzyl-p-toluenesulfonamide

To a suspension of P-HOBT 6 (1.01 g, 0.49 mmol) in CH₂Cl₂ (5 mL) wasadded p-toluenesulfonyl chloride (0.280 g, 1.47 mmol, 3.00 equiv.) andpyridine (0.155 g, 0.160 mL, 1.98 mmol, 4.04 equiv.). The suspension wassubsequently rocked for 60 minutes at 25° C. The polymer was thenfiltered and washed with CH₂Cl₂ (3×5 mL), MeCN (3×5 mL), and MeOH (3×5mL). The polymer was re-suspended in CH₂Cl₂ (5 mL), followed by theaddition of benzylamine (0.026 g, 0.027 mL, 0.24 mmol, 0.49 equiv. basedon P-HOBT 6). The suspension was rocked at 25° C. for 2 h. The polymerwas then filtered and washed with CH₂Cl₂ (2×5 mL). The filtrate andwashings were combined and concentrated to yield 0.050 g (77%) ofN-benzyl-p-toluenesulfonamide as a light yellow crystalline solid.

¹H NMR (300 MHz, CDCl₃): δ 2.44 (s, 3H, CH3), 4.14 (s, 2H, ArCH ₂), 4.60(bs, 1H, NH), 7.25 (m, 7H, ArH), 7.76 (m, 2H, ArH).

5. Acetylation of H₂N-MVTTD-COOH

AcHN-MVTTD-COOH

To a suspension of P-HOBT 3 (0.505 g, 0.19 mmol) in CH₂Cl₂ (5 mL) wasadded acetic anhydride (0.216 g, 0.200 mL, 2.12 mmol, 11 equiv.) andpyridine (0.234 g, 0.240 mL, 2.96 mmol, 16 equiv.). The suspension wassubsequently rocked for 60 minutes at 25° C. The polymer was thenfiltered and washed with CH₂Cl₂ (2×5 mL), MeOH (3×5 mL), DMF (3×5 mL),and MeOH (3×5 mL). The polymer was re-suspended in phosphate buffer(0.05 M, pH 7.0, 5 mL), followed by the addition of H₂N-MVTTD-COOH TFAsalt (prepared via standard solid phase peptide synthesis techniques,0.0020 g, 0.0029 mmol, 0.015 equiv. based on P-HOBT 3). The suspensionwas rocked at 25° C. for 5 h. The polymer was then filtered and washedwith water (5 mL). The filtrate and washings were combined andlyophilized. RP-HPLC [Waters binary gradient chromatography system,Phenomenex Jupiter C₁₈ semi-prep reversed-phase column (10 mm×250 mm,300Å pore size, 5 m particle size, Phenomenex, Torrance, Calif., 30° C.,3 mL/min, 220 nm, 0-37.5% MeCN (0.1% TFA) over 20 min] of the reactionmixture indicated a 50% conversion of H₂N-MVTTD-COOH (retention time12.8 min) to AcHN-MVTTD-COOH (retention time 15.7 min) with no otherproducts present.

6. Fish and Meat Packaging Sensor for Detection of Spoilage

As raw fish and meat products spoil due to improper handling or age,chemically reactive amines are produced in relatively highconcentrations. In particular, fish of the Scrombridae family (e.g.,tuna, mackeral) and non-scromboid relatives (e.g., bluefish, mahi-mahi,amberjack) release histamine during bacterial degradation (i.e.,spoilage). It is known that histamine causes scromboid poisoning, a formof food poisoning with severe symptoms, sometimes even death. Thecurrently accepted FDA amine detection method in fish is based onorganoleptic techniques (i.e., the use of smell). However, polymer bound1-hydroxybenzotriazole-6-carboxylic acid can be used to detect thepresence of amines with high sensitivity using absorbance (i.e., visiblecolor change) or fluorescence detection methods.

In a preferred embodiment of the present invention, the polymer bound1-hydroxybenzotriazole-6-carboxylic acid is utilized in the preparationof a disposable sensor for the detection of spoilage in packaged rawfish, meat, and poultry for consumer applications. The sensor includes athin circular wafer which is placed in direct contact with the food ofinterest before the final wrapping is put in place (See FIG. 7). In theabsence of amines, the wafer remains white, indicating that the food issafe to consume (see FIG. 6 a). However, when amines are present, theyreact with the immobilized 1-hydroxybenzotriazole-6-carboxylic acid,thereby releasing an FDA approved dye which then becomes visible on thesurface of the wafer. This color change indicates that the food is nolonger safe for consumption (see FIG. 6 b).

The interface with the raw fish or meat product is a porous membranehaving a pre-determined molecular weight cutoff (MWCO) which permitssmall molecules such as amines to pass, but will not allow intact cells,proteins, or other contaminants to pass. Suitable examples of the porousmembrane include cellulose, cellulose acetate, PVDF, polypropylene,polyurethane, polyacrylonitrile, nitrocellulose, polysulfone,polyacrylamide, polymethacrylate, polyamide and modified derivativesthereof. A second layer, which is located adjacent to the porousmembrane, is formed of a porous support to which1-hydroxybenzotriazole-6-carboxylic acid is via amide bond formation onan amine modified support, or bound via an ester linkage to a freehydroxyl group. Suitable examples of supports include cellulose,polypropylene, polyurethane, chitosan, polyacrylonitrile, polysulfone,polyvinyl alcohol, agarose, sepharose, polymethacrylate, polyacrylamide,polystyrene, polystyrene/polyethylene glycol graft copolymers, silicagels, glass beads, controlled pore glass and modified derivativesthereof.

The support may also include a hydroxyl group, a primary amine group ora secondary amine group. Once immobilized, the HOBT derivative ismodified with an FDA approved food dye (e.g., FD&C Red 3) viacarbodiimide mediated ester formation to yield the activated reagent asshown in FIG. 8. Other dyes, including but not limited to, the azo dyesDABCYL and DABSYL, may be utilized in the present invention.

The wafer is completed by the addition of a suitable diffusable layerwhich may be formed of polyvinylpyrrolidone, cellulose, celluloseacetate, PVDF, polypropylene, polyurethane, polyacrylonitrile,nitrocellulose, chitosan, polyvinylalcohol, polysulfone,polymethacrylate, polyacrylamide, polyamides and modified derivativesthereof. Other suitable examples would be easily determined by one ofskill in the art. This layer serves as the visible surface of thedevice, as well as for the potential entrapment of co-reagents (e.g., tomodify the environment in the sensor, such as pH) if necessary. Anon-porous, opaque top layer, e.g., polyamide (e.g., Nylon), PVC,polystyrene, polypropylene, polyethylene, polymethylmethacrylate orpolyester and modified derivatives thereof, is utilized as a mask toyield the design as described in FIG. 6 b. Once assembled, the waferdevice is held together by sealing the perimeter with an FDA approvedadhesive. After placement on the raw fish or meat surface, packagingmaterial is placed over the wafer to hold the wafer in place on thesurface of the meat product by pressure. Alternatively, an adhesive maybe applied to the outer perimeter of the top exposed surface of thewafer to adhere it to the packaging material.

Once in place, the wafer will function as shown in FIGS. 9 a and 9 b. Inparticular, when the meat is fresh, no amines are diffused through thecellulose acetate membrane. Consequently, no color is visible on thesurface of the wafer, as shown in FIG. 9 a. When the meat decomposes andspoils, amines are released. As shown in FIG. 9 b, the amines passthrough the porous MWCO membrane and react with the bound1-hydroxybenzotriazole-6-carboxylic acid/dye conjugate, therebyreleasing the modified dye. The modified dye then passes through thediffusable visible layer where color becomes visible in any unmaskedareas. The intensity of the color change is proportional to the totalamine concentration in the sample. Thus, a darker and more visible colorindicates a more spoiled meat product.

7. Amine Diagnostic Kit for Detection of Food Spoilage

In another preferred embodiment, the polymer bound1-hydroxybenzotriazole-6-carboxylic acid may be utilized for thedevelopment of diagnostic reagents or kits which can be used both onsite (qualitative) and off-site (quantitative) for the detection ofamines from food spoilage in wholesale environments (e.g., packingplants), retail environments, or for consumer applications. Thediagnostic kit (see FIG. 11) includes a transparent reaction chamber, acolor chart, packets of dye labeling agent, and an analysis buffer. Thetransparent reaction chamber is supplied with a snug fitting cap made ofrigid plastic, including but not limited to polymethylmethacrylate(PMMA), polystyrene, or polyethylene. In a preferred embodiment, the kitis contained in a housing. The housing (see FIG. 11) can be molded ormachined from a rigid plastic, including but not limited topolymethylmethacrylate (PMMA), polystyrene, and polyethylene. In afurther preferred embodiment, the kit includes a “sample kit” which isused to obtain the sample of interest. The sample kit includes itemssuch as cotton swabs, cellulose swipes, or filter paper swipes in adiscrete package.

The dye labeling reagent may be in the form of “tea bag” or “teststrip”. For the “tea bag”, 1-hydroxybenzotriazole-6-carboxylic acid isimmobilized on a solid support in bead or powder form via amide bondformation on an amine modified support, or bound via an ester linkage toa free hydroxyl group. The “tea bag” is of suitable dimensions such thatwhen it is filled with reagent it fits easily into the reaction chamber.The support includes, but is not limited to polystyrene,polystyrene/polyethylene glycol graft copolymers, silica gels, glassbeads, controlled pore glass, agarose, sepharose, cellulose, chitosan,polyacrylonitrile, polyurethane, polypropylene, polyvinyl alcohol,polysulfone, polymethacrylate, polyacrylamide and modified derivativesthereof. Additionally, the support may contain a hydroxyl group, aprimary amine group or a secondary amine group. Once immobilized, theHOBT derivative is modified with an FDA approved food dye (e.g., FD&CRed 3) via carbodiimide mediated ester formation to yield the activatedreagent. Other dyes, including but not limited to, the azo dyes DABCYLand DABSYL, may also be utilized. The immobilized1-hydroxybenzotriazole-6-carboxylic acid/dye conjugate is contained inthe “tea bag”. The “tea bag” material may be formed of filter paper,Nylon®, polyester, or cellulose. Other suitable examples will be easilydetermined by one of skill in the art. A single “tea bag” will beutilized for each analysis.

For the test strip dye labeling reagent,1-hydroxybenzotriazole-6-carboxylic acid is immobilized on a rigid,porous solid support (i.e., the strip) via amide bond formation on anamine modified support, or bound via an ester linkage to a free hydroxylgroup. The test strip is of suitable dimension such that when it isfilled with reagent, it fits easily into the reaction chamber. Suitablesupports include cellulose, polypropylene, polyurethane, chitosan,polyacrylonitrile, polysulfone, polyvinylalcohol, polyamide and modifiedderivatives thereof. Additionally, the support may contain a hydroxylgroup, a primary amine group, or a secondary amine group. Onceimmobilized, the HOBT derivative is modified with an FDA approved fooddye (e.g., FD&C Red 3) via carbodiimide mediated ester formation toyield the activated reagent. Other dyes, including but not limited tothe azo dyes DABCYL and DABSYL, may be utilized. A single “test strip”will be utilized for each analysis.

The buffer for use in the kits according to the present invention may beindividually or bulk prepackaged in powder form (dissolved in waterbefore use) or in bulk solution. The buffer includes phosphate,carbonate, acetate, HEPES, Tris, MES, or combinations thereof. Otherexamples can be easily determined by those of skill in the art.Modifiers, such as salts and chelating agents may be added.

In use, a sample of the food of interest is obtained and added to thereaction chamber along with a packet of dye labeling agent andappropriate amount of buffer. The reaction chamber is then closed andagitated for a finite period of time, after which time the dye labelingreagent is removed. The color of the resulting solution indicates degreeof spoilage (i.e., total amine content) of the food. A sample may betaken from the solution for further quantitative analysis, such as byany known analytical methods, to determine the identity andconcentration of the amines present in the food sample.

A specific usage of the amine diagnostic kit is described hereafter. Forexample, a sample of the food of interest (e.g., tuna fish) is obtainedby swiping the food surface with a cotton swab. The swab is then dippedinto the reagent chamber containing the buffer. The sample diffuses intobuffer from the swab. The swab is removed and a “tea bag” or “teststrip” containing the immobilized HOBT is added to the reaction chamber.The reaction chamber is then closed and agitated for two minutes, afterwhich the “tea bag” or “test strip” is removed. The color of theresulting solution indicates degree of spoilage (i.e., total aminecontent) of the food. In particular, the intensity of the color changeis proportional to the total amine concentration in the sample, and isdetermined by comparison to the color chart. Thus, a darker and morevisible color indicates a more spoiled food sample. A sample may betaken from the solution for further quantitative analysis, such as byknown analytical methods, to determine the identity and concentration ofthe amines present in the food sample.

The invention of this application is described above both generically,and with regard to specific embodiments. A wide variety of alternativesknown to those of ordinary skill in the art can be selected within thegeneric disclosure, and examples are not to be interpreted as limiting,unless specifically so indicated. The invention is not otherwiselimited, except for the recitation of the claims set forth below. Allreferences cited herein are incorporated in their entirety.

REFERENCES

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1-74. (canceled)
 75. An indicator for detecting the presence of an aminein a sample comprising an indicator releaseably bound to a solid ormembrane support; wherein in the presence of an amine, said indicator isreleased from said support, thereby causing a detectable color change;and wherein said color change is proportional to the amount of aminepresent in said sample.
 76. The indicator of claim 75, wherein saidindicator is releaseably bound to a compound linked to a solid ormembrane support, wherein said compound linked to said support isrepresented by the following formula:

wherein X represents a divalent linker moiety; R represents said solidor membrane support; and R′ represents a mono—or polyaromatic ring,which may include one or more heteroatoms.
 77. The indicator of claim76, wherein R′ is selected from the group consisting of1-hydroxybenzotriazole-6-carboxylic acid,2,3,5,6-tetrafluoro-4-hydroxybenzoic acid, 4-hydroxy-3-nitrobenzoicacid, 6-hydroxynicotinic acid, and2-hydroxy-1,3-dioxoisoindole-5-carboxylic acid, and derivatives thereof.78. The indicator of claim 75, wherein said support is selected from thegroup consisting of polystyrene, polystyrene/polyethylene glycol graftcopolymers, silica gels, glass beads, controlled pore glass, agarose,sepharose, cellulose, chitosan, polyacrylonitrile, polyurethane,polypropylene, polyvinyl alcohol, polymethacrylate, polyacrylamide,polysulfone and modified derivatives thereof.
 79. The indicator of claim76, wherein said linker moiety is selected from the group consisting ofN—R″ and O; wherein R″ is H or an alkyl group.
 80. The indicator ofclaim 76, wherein said compound linked to said support is1-hydroxybenzotriazole-6-carboxylic acid and is represented by thefollowing formula:

wherein R and X are as previously described.
 81. The indicator of claim75, wherein said releasable indicator is a dye.
 82. The indicator ofclaim 79, wherein said dye is FD&C Red
 3. 83. The indicator of claim 81,wherein said releasable indicator is an azo dye.
 84. The indicator ofclaim 83, wherein said azo dye is selected from the group consisting ofDABCYL and DABSYL.
 85. A compound linked to a solid or membrane support,wherein said compound linked to said support is represented by thefollowing formula:

R represents said support.
 86. The compound of claim 85, wherein R′ isselected from the group consisting of1-hydroxybenzotriazole-6-carboxylic acid,2,3,5,6-tetrafluoro-4-hydroxybenzoic acid, 4-hydroxy-3-nitrobenzoicacid, 6-hydroxynicotinic acid and2-hydroxy-1,3-dioxoisoindole-5-carboxylic acid, and derivatives thereof.87. The compound of claim 85, wherein said compound linked to saidsupport is 1-hydroxybenzotriazole-6-carboxylic acid and is representedby the following formula:

wherein R and X are as defined above.
 88. The compound of claim 85,wherein said support is selected from the group consisting ofpolystyrene, polystyrene/polyethylene glycol graft copolymers, silicagels, glass beads, controlled pore glass, agarose, sepharose, cellulose,chitosan, polyacrylonitrile, polyurethane, polypropylene, polyvinylalcohol, polymethacrylate, polyacrylamide, polysulfone and modifiedderivatives thereof.
 89. The compound of claim 85, wherein said linkermoiety is selected from the group consisting of N—R″ and O; wherein R″is H or an alkyl group.
 90. The compound of claim 85, wherein saidcompound bears a detectable agent selected from the group consisting ofa chromophoric agent, a fluorogenic agent, a radioactive agent and anelectrochemical agent.
 91. The compound of claim 90, wherein saiddetectable agent is selected from the group consisting of FD&C Red 3,DABCYL, DABSYL, pyrene, fluorescein, lucifer yellow, BODIPY, rhodamine,DANSYL, EDANS, .sup. 3H, .sup. 14C, .sup. 125I, .sup. 35S, .sup. 32P.92. The compound of claim 90, wherein the electrochemical agent containsa thiol or catechol moiety.
 93. The compound of claim 90, wherein saidamide or ester linkage enhances the chemical reactivity of saidcompound.